rabbit anti eno1 pab  (Proteintech)

Journal: Cell Reports Medicine

Article Title: Ferroptotic tumor cells reprogram tumor-associated macrophage antigen presentation to enhance the efficacy of immune checkpoint blockade

doi: 10.1016/j.xcrm.2026.102774

Figure Lengend Snippet: Ferroptosis signature associates with better responses to immunotherapy (A) Volcano plot analysis of differentially expressed genes from RNA-seq data of RSL3- and vehicle-treated MC38-bearing mice. (B) Correlation between ferroptosis signature score and cytotoxic T cell (CTL) signature score and CD38 expression in patients across solid malignancies. The Spearman correlation coefficient and associated probability were determined. All the data were obtained from TCGA. (C) Ferroptosis signature is positively correlated with CTL signatures and CD38 expression in most clinical trials for immunotherapy. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; Spearman correlation. (D) Ferroptosis signature score is higher in patients from the Riaz 2017 cohort evaluated as responders than in those evaluated as nonresponders. Data are presented as mean ± SD. ∗ p < 0.05; two-sided unpaired Student’s t test. (E) Ferroptosis signature score is higher in patients from Liu 2019 cohort evaluated as responders than in those evaluated as nonresponders. Data are presented as mean ± SD. ∗ p < 0.05; two-sided unpaired Student’s t test. (F) Patients with higher ferroptosis signature score have better progress-free survival in patients from the Liu 2019 cohort treated with anti-PD-1; log rank (Mantel-Cox) test. (G and H) Representative multiplex immunofluorescence images of TFRC, IDO1, ENO1, and ACSL4 (G) were shown from biopsy samples of responders ( n = 6) or non-responders ( n = 6) to immunotherapy in CRC, and the fluorescence signals were quantified (H). Scale bar: 50 μM. Data are presented as mean ± SD. ∗∗∗ p < 0.001; two-sided unpaired Student’s t test. (I) Representative multiplex immunofluorescence images of CD68 and MHC-II co-location from biopsy samples of responders ( n = 6) or non-responders ( n = 6) to immunotherapy in CRC. Scale bar: 50 μM. (J) Correlation between the number of MHC-II + CD68 + macrophages and ferroptosis signature score. Spearman correlation. (K) Representative positron emission tomography-computed tomography (PET-CT) of patients receiving anti-PD-1 antibody treatment. (L) Representative CT of patients receiving anti-PD-1 antibody treatment.

Article Snippet: Rabbit polyclonal anti-human/mouse ENO1 antibody , ABclonal , Cat# A1033; RRID: AB_2757874.

Techniques: RNA Sequencing, Expressing, Clinical Proteomics, Multiplex Assay, Immunofluorescence, Fluorescence, Positron Emission Tomography, Computed Tomography, Positron Emission Tomography-Computed Tomography

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex

doi: 10.1007/s00018-026-06146-9

Figure Lengend Snippet: ENO1 interacts with the NP protein. ( A , B ) HEK293T cells were transfected individually or in combination with Flag-tagged ENO1 and HA-tagged NP. At 24 h post-transfection, lysates of cells were immunoprecipitated with mouse anti-Flag mAb (A) or mouse anti-HA mAb (B) and then western blotted with rabbit anti-Flag pAb and rabbit anti-HA pAb, respectively. ( C ) HEK293T cells were infected with H1N1 virus (MOI = 5). At 12 h p.i., lysates from HEK293T cells were immunoprecipitated with mouse anti-ENO1 pAb, followed by western blotting with rabbit anti-ENO1 pAb and rabbit anti-NP pAb, respectively. ( D ) Purified GST and GST-ENO1 protein were used to pull down Flag-tagged NP. ( E ) A549 cells were uninfected or infected with H1N1 virus (MOI = 5). At 12 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 5 μm. ( F , G ) HEK293T cells were transfected with the indicated plasmids. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-Flag mAb, followed by western blotting with rabbit anti-Flag pAb, rabbit anti-HA pAb, and rabbit anti-V5 pAb. The data are representative of at least three independent experiments

Article Snippet: The following primary antibodies were obtained from commercial sources: mouse anti-Flag mAb (A00187 -100, GenScript), rabbit anti-Flag pAb (20543-1-AP, Proteintech) mouse anti-HA mAb (A01244-100, GenScript), rabbit anti-HA pAb (51064-2-AP, Proteintech), mouse anti-V5 mAb (A01724-100, GenScript), rabbit anti-V5 pAb (14440-1-AP, Proteintech), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech), rabbit anti-ENO1 pAb (11204-1-AP, Proteintech), rabbit anti-LaminA/C pAb (10298-1-AP, Proteintech), rabbit anti-KPNA4 pAb (12463-1-AP, Proteintech), rabbit anti-PB2 pAb (GTX125926, GeneTex), rabbit anti-PB1 pAb (GTX125923, GeneTex), rabbit anti-PA pAb (GTX118991, GeneTex), rabbit anti-NP pAb (GTX125989, GeneTex).

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Virus, Purification, Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex

doi: 10.1007/s00018-026-06146-9

Figure Lengend Snippet: ENO1 impairs the nuclear import of the vRNP complex of IAV. ( A ) HEK293T cells were transfected with HA-tagged NP and Flag-tagged NP, together with Flag-tagged ENO1 or not. At 24 h post-transfection, cell lysates were immunoprecipitated with mouse anti-HA mAb and subjected to western blotting. ( B ) The effect of ENO1 on the vRNP assembly was determined using a co-IP assay. HEK293T cells were transfected with plasmids expressing vRNP proteins (PB2, PB1, PA, NP) and pPol I, together with Flag-tagged ENO1. Cell lysates were immunoprecipitated with mouse anti-HA antibody, followed by western blotting. ( C ) Si-ENO1- or NC-treated A549 cells were infected with H1N1 virus (MOI = 5). At 3 h p.i., the cells were separated into nuclear and cytoplasmic fractions, and each fraction was then western blotted with rabbit anti-ENO1, NP, GAPDH, and Lamin A/C pAb. The intensities of the NP protein bands were determined by using lmageJ, and the ratio of nuclear NP were normalized to cytoplasmic NP. ( D ) A549 cells transfected with Flag-tagged ENO1 or empty vector were infected with H1N1 virus (MOI = 5). At 3 h p.i., the cells were separated into nuclear and cytoplasmic fractions, and each fraction was then western blotted with rabbit anti-ENO1, NP, GAPDH, and Lamin A/C pAb. The intensities of the NP protein bands were determined by using lmageJ, and the ratio of nuclear NP were normalized to cytoplasmic NP. ( E ) A549 cells transfected with Flag-tagged ENO1 or empty vector were infected with H1N1 virus (MOI = 5). At 3 h p.i., the cells were fixed and stained with mouse anti-NP mAb and rabbit anti-ENO1 pAb. Scale bars, 20 μm. The data are representative of at least three independent experiments

Article Snippet: The following primary antibodies were obtained from commercial sources: mouse anti-Flag mAb (A00187 -100, GenScript), rabbit anti-Flag pAb (20543-1-AP, Proteintech) mouse anti-HA mAb (A01244-100, GenScript), rabbit anti-HA pAb (51064-2-AP, Proteintech), mouse anti-V5 mAb (A01724-100, GenScript), rabbit anti-V5 pAb (14440-1-AP, Proteintech), rabbit anti-GAPDH pAb (10494-1-AP, Proteintech), rabbit anti-ENO1 pAb (11204-1-AP, Proteintech), rabbit anti-LaminA/C pAb (10298-1-AP, Proteintech), rabbit anti-KPNA4 pAb (12463-1-AP, Proteintech), rabbit anti-PB2 pAb (GTX125926, GeneTex), rabbit anti-PB1 pAb (GTX125923, GeneTex), rabbit anti-PA pAb (GTX118991, GeneTex), rabbit anti-NP pAb (GTX125989, GeneTex).

Techniques: Transfection, Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay, Expressing, Infection, Virus, Plasmid Preparation, Staining

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Secretome profiling via 4D-FastDIA proteomics and GeneCards database screening identified secreted proteins. B Heatmap analysis of the identified secreted proteins. C Immunofluorescence (IF) staining demonstrating ENO1 colocalization with autophagy marker LC3B. Scale bars, 20 μm. D Western blot and ELISA analysis of secreted ENO1 levels in conditioned media from GBM cell lines exposed to TMZ (0–1000 μM, 48 h). E Time-course analysis (fixed 600 μM TMZ) monitoring ENO1 secretion by Western blot and ELISA. F Extracellular ENO1 quantification by ELISA and membrane integrity assessment via LDH release assay in 600 μM TMZ-treated models. G Co-immunoprecipitation (Co-IP) of GBM cell lysates using anti-ENO1 antibody, followed by immunoblotting with anti-TRIM16 and anti-SEC22B antibodies. H Western blot analysis of GAL3 and GAL8 expression in GBM cells treated with 600 μM TMZ. I IF double-labeling showing LLOMe (0.5 μM)-induced subcellular colocalization of LC3B and TRIM16. Scale bars, 20 μm. J Dose-response analysis: GBM cells treated with LLOMe (0, 0.25, 0.5, 1 μM; 24 h). Intracellular ENO1, LC3B-I-to-II conversion (Western blot), and secreted ENO1 (ELISA) were concurrently measured. K Time-course analysis (fixed 1 μM LLOMe; 0, 6, 12, 24 h): Intracellular ENO1/LC3B (Western blot) and secreted ENO1 (ELISA). L ATG5-knockdown validation: shNC/shATG5-transfected GBM cells ± TMZ (600 μM, 24 h). Immunoblotting for ATG5, secreted ENO1, and LC3B lipidation; secreted ENO1 quantified by ELISA. M Bafilomycin A1 (BafA1, 200 nM, 6 h pre-treatment) ± TMZ (600 μM, 24 h). Immunoblotting of whole-cell lysates (WCL) and conditioned media (CM) for LC3B/ENO1; secreted ENO1 by ELISA. N 3-Methyladenine (3-MA, 5 mM, 6 h pre-treatment) ± TMZ (600 μM, 24 h). Immunoblotting of WCL and CM for LC3B/ENO1; secreted ENO1 by ELISA. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Enzyme-linked Immunosorbent Assay, Membrane, Lactate Dehydrogenase Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Labeling, Knockdown, Biomarker Discovery, Transfection

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Pan-cancer analysis of ENO1 expression. ENO1 expression in normal brain tissues and GBM tumor tissues at mRNA ( B ) and protein levels ( C ). D Heatmap analysis correlating ENO1 expression with clinicopathological features in glioma patients. Significant associations between ENO1 expression and WHO grading ( E ) or histopathological subtypes ( F ). G Kaplan-Meier overall survival (OS) analysis of TCGA-GBM patients stratified by high/low ENO1 mRNA expression. H Analysis of mRNA expression levels of ENO1 in patients with primary recurrent gliomas based on the CGGA database. I Overall survival (OS) analysis of TMZ-treated glioma patients stratified by ENO1 expression based on the CGGA-LGG + GBM database. J Representative IHC images and quantitative scores of ENO1 in paired primary/recurrent GBM specimens pre- and post-TMZ therapy. Scale bars, 50 μm. K Serum ENO1 levels quantified by ELISA across glioma WHO grades. L ELISA quantitative analysis of serum ENO1 concentrations in GBM patients pre- and post-TMZ chemotherapy. M TMZ sensitivity assays following exogenous rhENO1 (1 μg/mL) administration, measured by IC50 shifts. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Western blot analysis of ENO1 knockdown efficiency in both intracellular and extracellular fractions of U87MG cells. B Western blot validation of ENO1 overexpression in LN229 cells and conditioned media (CM). Colony formation ( C ) and EdU proliferation ( D ) assays of parental LN229 cells treated with CM from ENO1-overexpressing LN229 cells. Scale bars, 100 μm. E , F Metastatic potential assessment using CM from ENO1-overexpressing cells: E Wound healing assay (scale bar = 500 μm). F Transwell migration and invasion assays (scale bar = 100 μm). G Western blot detection of intracellular ENO1 after 24 h treatment with recombinant human ENO1 (rhENO1, 1 μg/mL). H CCK-8 assay evaluating proliferation in rhENO1-treated cells (1 μg/mL). I Transwell migration/invasion of rhENO1-treated GBM cells (scale bars, 100 μm). J Scratch wound healing assay post-rhENO1 treatment (scale bars, 500 μm). Rescue experiments: Proliferation ( K , L ) and migration/invasion ( M , N ) capacities of GBM cells treated with shENO1 CM supplemented with rhENO1. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: Western Blot, Knockdown, Biomarker Discovery, Over Expression, Wound Healing Assay, Migration, Recombinant, CCK-8 Assay

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Immunoprecipitation-mass spectrometry (IP-MS) analysis of ENO1-interacting proteins in U87MG cells treated with rhENO1 (0-12 h) using ENO1-conjugated beads, with whole cell protein (WCP) and IgG as controls (silver staining shown). B Identification of ENO1-binding transmembrane proteins (TMPs) from 70-130 kDa differential bands by MS, screened against the Membranome database (containing 2364 TMPs). C Molecular docking model of ENO1-TLR4 interaction (ENO1 shown in deep blue, TLR4 in cyan; binding sites displayed as stick structures in corresponding colors). D – F Co-immunoprecipitation (Co-IP) validation of ENO1-TLR4 interaction in GBM cells: D Co-IP validated that ENO1 binds to TLR4 in shENO1 GBM cells. E Co-IP with anti-ENO1 antibody followed by immunoblotting of rhENO1-treated GBM cells with anti-ENO1 and anti-TLR4 antibodies. F Co-IP with anti-TLR4 antibody followed by immunoblotting of rhENO1-treated GBM cells with anti-ENO1 and anti-TLR4 antibodies. G Immunofluorescence analysis of ENO1 (green) and TLR4 (red) colocalization in rhENO1-stimulated GBM cells and TMZ-treated human GBM specimens. Scale bar = 10 μm. H Pearson correlation analysis of ENO1 and TLR4 mRNA expression in TCGA-GBM and CGGA-GBM datasets. Representative images and quantitative analysis of ( I ) proliferation, ( J ) colony formation, and migration/invasion ( K , L ) in GBM cells treated with or without TLR4 inhibition and cultured in rhENO1. Scale bars = 5 mm ( J ), 100 μm ( I , K ), and 500 μm ( L ). Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Silver Staining, Binding Assay, Co-Immunoprecipitation Assay, Biomarker Discovery, Western Blot, Immunofluorescence, Expressing, Migration, Inhibition, Cell Culture

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Cluster analysis of differentially expressed genes (DEGs) between rhENO1-treated and control groups (n = 3 biological replicates). B Volcano plot of DEGs displaying: X-axis: log2 fold-change in gene expression; Y-axis: -log10(p-value) for statistical significance; Green dots: non-significant genes; Red/blue dots: significantly up-/down-regulated genes. C KEGG pathway analysis revealing significant association between ENO1 and PI3K- Akt signaling in GBM patient samples. D GSEA enrichment plot demonstrating PI3K- Akt pathway activation correlated with ENO1 expression from the TCGA database. E Western blot analysis of PI3K and Akt phosphorylation in rhENO1-treated GBM cells using anti-phospho-PI3K, anti-PI3K, anti-phospho- Akt, and anti- Akt antibodies. F Western blot analysis of PI3K/Akt phosphorylation in serum-starved GBM cells treated with/without rhENO1 (1 μg/mL) and PI3K inhibitor LY294002. Representative images and quantitative analysis of G colony formation, H proliferation, and migration/invasion I , J in GBM cells treated with or without PI3K inhibition LY294002 and cultured in rhENO1. Scale bars = 5 mm ( G ), 100 μm ( H, J ), and 500 μm ( I ). K Western blot analysis of PI3K/Akt phosphorylation in serum-starved GBM cells treated with/without rhENO1 (1 μg/mL) and TLR4 inhibitor TAK242 (10 μM). Data represent mean ± SEM; ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: Control, Gene Expression, Activation Assay, Expressing, Western Blot, Phospho-proteomics, Migration, Inhibition, Cell Culture

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Principal component analysis (PCA) of metabolites and lipids detected by untargeted lipidomics in GBM cells treated with soluble recombinant ENO1 (rhENO1, 1 μg/mL) (n = 6 biological replicates). B KEGG pathway enrichment analysis of differential metabolites with VIP > 1. C Heatmap analysis of differential metabolites with VIP > 1 (n = 6 biological replicates). D Sphingomyelin (SM) was significantly upregulated in rhENO1-treated samples. E Western blot analysis of SPHK1 and ERK phosphorylation levels in serum-starved GBM cells treated with or without rhENO1 (1 μg/mL) for 6 h. F Western blot analysis using anti-phospho-SPHK1, anti-SPHK1, anti-phospho-ERK, and anti-ERK antibodies in serum-starved GBM cells treated with or without rhENO1 (1 μg/mL) and TLR4 inhibitor (TAK242, 10 μM) for 6 h. G Immunofluorescence staining of SPHK1 subcellular localization in GBM cells treated with rhENO1 (1 μg/mL) and TLR4 inhibitor (TAK242, 10 μM). Scale bar = 10 μm. H ELISA quantification of S1P levels in conditioned medium from rhENO1-treated GBM cells. I Western blot analysis of SPHK1 phosphorylation levels in GBM cells treated with increasing concentrations of SPHK1 inhibitor PF-543. J-M Representative images and quantification of J proliferation (scale bar = 100 μm), K colony formation (scale bar = 5 mm), L migration (scale bar = 100 μm), and M invasion (scale bar = 500 μm) in GBM cells treated with DMSO control, PF-543 (10 μM), or S1P (1 μM). Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: Recombinant, Western Blot, Phospho-proteomics, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Migration, Control

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Dimensionality reduction and clustering of single-cell RNA sequencing (scRNA-seq) and spatial transcriptomic RNA sequencing (stRNA-seq) data from GEO database. B Cluster analysis of peritumoral and tumor core regions in GBM specimens. C MIA (Microenvironment Cell Populations) analysis evaluating cell type enrichment across different clusters in tumor core versus peritumoral regions. D Spatial distribution patterns of ENO1 expression in GBM tumor core and peritumoral regions. E qRT-PCR quantification of M2-TAM markers (CD163, CD206, ARG1, IL-10) mRNA levels following rhENO1 treatment (1 μg/mL). F Immunofluorescence analysis of M2-TAM markers (CD68, CD163, CD206) subcellular localization and expression intensity after rhENO1 treatment (1 μg/mL). Scale bar = 10 μm. G Immunofluorescence staining of M2-TAM markers (CD163, CD206) in TMZ-treated co-culture system of sh-ENO1 U87MG cells with M0 macrophages. Scale bar = 10 μm. H qRT-PCR analysis of M2-TAM markers (CD163, CD206, ARG1) mRNA expression in TMZ-treated sh-ENO1 U87MG/M0 macrophage co-culture system. I Immunohistochemistry (IHC) analysis of the correlation between CD163 and ENO1 in GBM tissues. J Confocal microscopy quantification of membrane localization intensity for M2-TAM surface markers (CD163/CD206) following treatment with rhENO1 (1 μg/mL) and SPHK1 inhibitor PF-543 (10 μM). Scale bar = 10 μm. K qRT-PCR detection of M2-TAM markers (CD163, CD206, ARG1) mRNA levels under combined rhENO1 (1 μg/mL) and PF-543 (10 μM) treatment. L The levels of cytokines IL-10, CCL18, and TGF-β released in the supernatant of THP-1-derived macrophages were detected by ELISA after indicated treatment for 24 h. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Co-Culture Assay, Immunohistochemistry, Confocal Microscopy, Membrane, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Autophagy-dependent secretion of ENO1 mediates chemoresistance of glioblastoma and tumor microenvironment remodeling

doi: 10.1038/s41419-025-08313-5

Figure Lengend Snippet: A Experimental workflow of orthotopic glioma mouse model and treatment regimen (n = 6). B Representative in vivo bioluminescence images of Luci-GL261-bearing mice treated with:(i) saline control, (ii) TMZ monotherapy, (iii) TMZ + PF-543 (SPHK1 inhibitor), (iv) TMZ + PF-543 + TAK242 (TLR4 inhibitor) at days 7, 14, and 21 post-implantation. C , D Quantitative analysis of tumor growth using relative flux values (normalized to day 7) across treatment groups. E Kaplan-Meier survival curves showing intracranial tumor progression in the four treatment cohorts; n = 6 mice. F Representative immunohistochemical staining of ENO1, p-SPHK1, and CD163 in tumor sections from each treatment group. Scale bar, 100 μm. G Schematic diagram depicting the mechanistic basis of ENO1 mediating cellular communication between GBM cells and M2-TAMs through an autophagy-dependent secretory pathway and driving TMZ chemoresistance. Data represent mean ± SEM; ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Antibodies used for target protein IP included: ENO1 Rabbit pAb (Proteintech, Cat# 11204-1-AP; 1:100 dilution), SEC22B Rabbit pAb (Proteintech, Cat# 14776-1-AP; 1:100 dilution), TRIM16 Rabbit pAb (Proteintech, Cat# 24403-1-AP; 1:100 dilution), TLR4 Mouse mAb (Abcam, Cat# ab22048; 1:100 dilution) and control IgG (Proteintech, Cat# 30000-0-AP; 1:100 dilution).

Techniques: In Vivo, Saline, Control, Immunohistochemical staining, Staining

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: Increased protein lactylation in the PECs of ARDS mice, with Eno1 as a substrate. (A) Histogram of the ratio distribution of quantifiable Klac sites in the PECs of control and LPS‐induced ARDS mice. (B) GO items associated with Klac sites (ARDS/Control > +∞) located proteins. (C) Venn diagram of predicted CXCL12 3′ UTR binding proteins and proteins in picked GO items and the average protein copies of 13 screened proteins in PECs. (D) Ribbon diagram of the crystal structure of mouse Eno1 and lactylation at the K193 residue. (E) Mass spectrometry of Eno1 lactylated at K193. (F) Purified mouse Eno1 (100 µg/mL) was incubated with different concentrations of lactyl‐CoA for 1 h at 37°C. Then, the mixtures were added to protein loading buffer for denaturation, and K‐lactylation was assessed by western blotting. (G) The K193 site in Eno1 is conserved in different species. Conserved K193 sites are marked in red in the sequences among different species. (H) Western blotting analysis of the K193 lactylated level in PECs isolated from the lungs of control and LPS‐induced ARDS mice. (I) In vitro, the K193 lactylation levels in PBS‐ and LPS‐activated MPMECs were measured by western blotting.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Control, Binding Assay, Residue, Mass Spectrometry, Purification, Incubation, Western Blot, Isolation, In Vitro

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: K193 lactylated Eno1 released the bound CXCL12 mRNA to accelerate translation. (A) Eno1 expression in MPMECs transfected with siRNAs. (B) CXCL12 mRNA and (C) protein expression in Eno1 KD cells after LPS stimulation ( n = 3). (D) Molecular docking and ΔG binding energy calculation (E) RIP‐qPCR detection of Eno1‐bound CXCL12 transcripts in MPMECs treated with LPS ( n = 3). (F) Click‐iT AHA assays to detect the CXCL12 translation efficacy in MPMECs treated with LPS ( n = 3). (G) Schematics of the reporter and effector constructs. Dual‐luciferase analysis in HEK293T cells transfected with the reporter expressing the CXCL12‐3′ UTR and constructs expressing the WT, K193Q, or K193R Eno1 ( n = 3). (H) CXCL12 concentrations in culture media from MPMECs transfected with shEno1, shEno1+oe‐WT, or shEno1+oe‐K193R ( n = 3). * p < 0.05, ** p < 0.01, **** p < 0.001.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Expressing, Transfection, Binding Assay, Construct, Luciferase

Journal: MedComm

Article Title: Global Lactylome Reveals Lactylation‐Dependent Mechanisms Underlying CXC Motif Chemokine Ligand 12 Expression in Pulmonary Endothelium During Acute Respiratory Distress Syndrome

doi: 10.1002/mco2.70344

Figure Lengend Snippet: K193 lactylation augmented Eno1 enzymatic activity. (A) The levels of lactate in the culture medium of PBS‐ and LPS‐treated MPMECs over time ( n = 3). (B) PBS‐ and LPS‐treated MPMECs lysates were prepared to measure Eno1 enzymatic activity by monitoring phosphoenolpyruvate concentrations over time ( n = 3). (C) Eno1 enzymatic activity was measured in lysates from shEno1+oe‐WT and shEno1+oe‐K193R MPMEC ( n = 6). * p < 0.05. In C, *compared with the untreated shEno1+oe‐WT group, * p < 0.05; # compared with the lactate‐treated shEno1+oe‐K193R group, # p < 0.05.

Article Snippet: The following primary antibodies were used: anti‐L‐lactyl lysine (PTM‐1401, PTM Biolabs, China), anti‐mouse Eno1 (A1033, ABclonal, China), anti‐K193 lactylated Eno1 (custom designed, ABclonal, China), and anti‐mouse β‐actin (ZF0033, ZFdows Bio, China).

Techniques: Activity Assay

Journal: Cell Death Discovery

Article Title: A novel protein encoded by circUBE2G1 suppresses glycolysis in gastric cancer through binding to ENO1

doi: 10.1038/s41420-025-02644-0

Figure Lengend Snippet: A In the left panel, Total protein from circUBE2G1-99aa plasmid-transfected HGC-27 cells was separated via SDS-PAGE. Arrows show different bands between IgG and Flag. ENO1 was identified by LC/LC-MS in circUBE2G1-99aa protein complex. Kyoto Encyclopedia of Genes B and Genomes pathway analysis C showed the significantly affected signaling pathway on circUBE2G1-99aa overexpression in GC cells. D Table presenting scores of glycolytic-related proteins identified by mass spectrometry to interact with circUBE2G1-99aa. E Illustration depicting the predicted binding mode of circUBE2G1-99aa with ENO1, as modeled using AlphaFold and visualized in PyMol software. F Mutual interaction of ENO1 and Flag-circUBE2G1-99aa was determined by IP. G Flag-tagged circUBE2G1-99aa was transfected into HGC-27 and AGS cells, and immunofluorescence was performed using anti-Flag and anti-ENO1 antibodies. Scale bar, 10 μm. H Overexpress circUBE2G1-99aa in GC cell lines HGC-27 and AGS, then quantify ENO1 mRNA and protein expression levels using qRT-PCR and Western blot, respectively. I Overexpression of circUBE2G1-99aa in GC cells HGC-27 and AGS, followed by measurement of ENO1 enzyme activity. J Left panel, Schematic illustration of ENO1 domain truncation and the circUBE2G1-99aa truncation diagram. Right panel, Co-IP validation of the binding region between circUBE2G1-99aa and ENO1. K Glucose consumption, pyruvate, lactate production, and ATP generation were measured in HGC-27 and AGS cells divided into three groups: Empty vector, circUBE2G1-99aa, and circUBE2G1-99aa + ENO1. L Extracellular acidification rates and cellular oxygen consumption rates were determined using the Seahorse assay for each group. Graph represents mean ± SD; ns not significant, ** P < 0.01, and *** P < 0.001.

Article Snippet: The product numbers of antibodies were listed: QKI (A22723, ABclonal, 1:1000), ENO1 (A1033, ABclonal, 1:1000), AKT (AP0059, Bioworld, 1:1000), p-AKT phosphorylated Ser473 (9271, Cell Signaling Technology, 1:1000), PI3K (ab70598, Abcam, 1:1000), p-PI3K phosphorylated Tyr607, β-tubulin (ab6046, Abcam, 1:500), GAPDH, and β-actin were used as internal controls.

Techniques: Plasmid Preparation, Transfection, SDS Page, Liquid Chromatography with Mass Spectroscopy, Over Expression, Mass Spectrometry, Binding Assay, Software, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Co-Immunoprecipitation Assay, Biomarker Discovery

Journal: Cell Death Discovery

Article Title: A novel protein encoded by circUBE2G1 suppresses glycolysis in gastric cancer through binding to ENO1

doi: 10.1038/s41420-025-02644-0

Figure Lengend Snippet: A An evaluation of PI3K/AKT signaling pathway-related protein level alterations by Western blot in HGC-27 and AGS cells stably overexpressing circUBE2G1-99aa following ENO1 overexpression. B Investigation of PI3K/AKT signaling pathway protein levels via Western blot in circUBE2G1-99aa-stably overexpressing HGC-27 and AGS cells treated with the PI3K/AKT pathway activator 740 Y-P. C , D Measurement of glucose consumption, pyruvate, lactate production, ATP levels, and ECAR and OCR using Seahorse analysis in HGC-27 and AGS cells divided into Empty vector, circUBE2G1-99aa, and circUBE2G1-99aa + 740 Y-P groups. E Subcutaneous xenograft tumor models established in BALB/c Nude mice using HGC-27 cell lines (Empty vector, circUBE2G1-OE, circUBE2G1-ATG mut, and Linear circUBE2G1-99aa), followed by H&E staining and immunohistochemical detection of p-PI3K (Tyr607) and p-AKT (Ser473) expression levels in tumor tissues. Scale bar: 100 μm. F Schematic illustration of the mechanism by which circUBE2G1-99aa regulates the ENO1/PI3K/AKT axis to inhibit glycolysis in GC cells. Graph represents mean ± SD; ns not significant, * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The product numbers of antibodies were listed: QKI (A22723, ABclonal, 1:1000), ENO1 (A1033, ABclonal, 1:1000), AKT (AP0059, Bioworld, 1:1000), p-AKT phosphorylated Ser473 (9271, Cell Signaling Technology, 1:1000), PI3K (ab70598, Abcam, 1:1000), p-PI3K phosphorylated Tyr607, β-tubulin (ab6046, Abcam, 1:500), GAPDH, and β-actin were used as internal controls.

Techniques: Western Blot, Stable Transfection, Over Expression, Plasmid Preparation, Staining, Immunohistochemical staining, Expressing